The C-terminus of phosphatidylinositol transfer protein modulates membrane interactions and transfer activity but not phospholipid binding.
Rat phosphatidylinositol transfer protein (PITP) is a 32 kDa protein containing 271 amino acids. It is involved in a number of cell functions including secretion and cell signaling. To further characterize structure/activity relationships of PITP, two C-terminal truncated derivatives, PITP(1-259) and PITP(1-253), were produced in Escherichia coli and purified to homogeneity. PITP(1-259) had transfer activity equal to 30-40% to that of native PITP in transfer of either phosphatidylcholine (PC) or phosphatidylinositol (PI) when transfer was measured using 95/5 mol% PC/PI donor and acceptor vesicles; PITP(1-253) had only slight transfer activity, even under the most favorable assay conditions. Thus, amino acids 254-258 are critical for transfer activity. The transfer activity of PITP(1-259) was strongly dependent on the composition of the donor and acceptor vesicles. With 100 mol% PC donor and acceptor vesicles, PITP(1-259) transfer activity ranged from 70 to 100% to that of PITP. The presence of 2 mol% phosphatidic acid (PA) in either donor or acceptor vesicles reduced transfer activity to between 10 and 20% that of full-length PITP under the same conditions. If both donor and acceptor contained 2% PA, PITP(1-259) was essentially inactive, though the activity of PITP was not affected significantly under these conditions. PITP(1-253) and PITP(1-259) bind much more avidly to vesicles than does PITP, and this enhanced binding reflects increased electrostatic interactions. Thus, the C-terminal residues modulate the affinity of PITP for vesicles and the efficiency of phospholipid transfer.
Tremblay
J M
JM
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160-7421, USA.
Voziyan
P A
PA
Helmkamp
G M
GM
Yarbrough
L R
LR
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010712:Phosphatidic Acids / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D002352:Carrier Proteins; D008081:Liposomes; D008565:Membrane Proteins; D010446:Peptide Fragments; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D011994:Recombinant Proteins